Method of stabilizing and potentiating the action of anti-angiogenic substances

ABSTRACT

A method of stabilizing and potentiating action of molecules of known anti-angiogenic substances such as ANGIOSTATIN® or ENDOSTATIN® by using in coupling conjugation with cis-unsaturated fatty acids (c-UFAs) in the treatment of cell proliferative disorders uses c-UFAs chosen from linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid and cis-parinaric acid in predetermined quantities. Preferably, the c-UFAs are in the form of polyunsaturated fatty acids (PUFAs). Uncontrolled or undesirable angiogenic activity promotes cell proliferative disorders and tumor growth, which can be inhibited by the selective use of PUFAs with anti-angiogenic substances used selectively in conjunction with predetermined anti-cancer drugs. For a non-glioma type of cell proliferation disorder, a sodium, potassium or lithium salt of a PUFA is preferred to form an admixture with an anti-angiogenic substance. Anti-angiogenic substances envisaged in this invention include ANGIOSTATIN, ENDOSTATIN, platelet factor-4, TNP-470, thalidomide, interleukin-12 and metalloproteinase inhibitors (MMP). A preferred method of administration of the mixture to treat a tumor is intra-arterial administration into an artery which provides the main blood supply for the tumor.

This application is a divisional of U.S. application Ser. No. 09/478,291filed on 5 Jan. 2000 now U.S. Pat. No. 6,380,253.

FIELD OF THE INVENTION

The present invention generally relates to the use of anti-angiogenicagents in the cure of cell proliferative disorders including cancer andother disorders caused by uncontrolled angiogenic activity in the body.More particularly, the invention is directed to the efficacious use ofanti-angiogenic agents.

RELATED APPLICATIONS

This invention relates to co-pending U.S. application Ser. No.09/392,953 filed on Sep. 9, 1999 and entitled “Method of Treatment forCell proliferative Disorders including Cancer”, which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The term angiogenesis refers to the generation or formation of new bloodvessels into a tissue or organ. Angiogenesis can occur both during somephysiological processes and/or in some pathological conditions. Forexample, angiogenesis can be seen to occur during wound healing, fetalgrowth, corpus luteum, and endometrium, etc., (1). Endothelial cells,which cause to form the inner lining of the blood vessels, areconstituted by a thin layer of epithelial cells and these cells arenecessary for the process of angiogenesis. During the process ofangiogenesis, irrespective of whether it is physiological orpathological, the endothelial cells release enzymes which can produceerosions of the basement membrane through which the endothelial cellscause protrusions. In response to the stimuli given by various agents,endothelial cells proliferate and migrate through the protrusions andform a sprout of the parent blood vessel. These endothelial cell sproutscan merge to form capillary loops leading to the formation of new bloodvessel(s). If the blood vessels are in a tumor area, these new bloodvessels in turn will provide enough nutrients and energy sources so thattumor cells can divide, proliferate and grow both in number and size.

Thus, the process of angiogenesis is both essential and critical to thegrowth of cancer. The other pathological states in which angiogenesisplays a critical role include: rheumatoid arthritis, psoriasis,scleroderma, myocardial angiogenesis, corneal diseases, diabeticretinopathy associated with neovascularization, macular degeneration,ovulation, menstruation etc. The process of angiogenesis also appears tobe critical for tumor metastasis.

Since angiogenesis is such a critical process in the promotion of cancerand tumor metastasis, several researches have been trying to devisemethods or develop drugs which can selectively suppress angiogenesiswith the hope that this would eventually lead to the inhibition of tumorgrowth. There are other situations where uncontrolled angiogenesis isundesirable. For instance, formation of new blood vessels in an arealike cornea during the process of healing of the corneal ulcer, if it isin excess, can lead to corneal scar formation.

In the case of rheumatoid arthritis, angiogenesis can lead to continuedinflammation in the joints and also to osteoporosis. In such aninstance, prevention of formation of new blood vessels will lead toreduction in inflammation and also prevention of fibrous ankylosis andbony ankylosis. Thus, selective prevention and control of angiogenesismay be of benefit in the aforementioned conditions, as well as inseveral other conditions such as: uterine fibroids, psoriasis,scleroderma, diabetic retinopathy, keloids, ovulation etc. Another areawhere prevention of angiogenesis will be of benefit is in the inhibitionof ovulation and menstruation and growth of placenta and this will leadto prevention of fertilization and growth of the fetal tissue. This may,thus, form a new approach in the development of fertility controlmeasures.

Two naturally occurring molecules which have been identified toadversely influence or inhibit angiogenesis are ANGIOSTATIN® andENDOSTATIN® (2). Both these molecules are proteins. ANGIOSTATIN is aprotein of molecular weight approximately 38 kD and has an amino acidsequence substantially similar to that of a fragment of murineplasminogen beginning at amino acid number 98 of an intact murineplasminogen molecule. The amino acid sequence of ANGIOSTATIN varies onlyslightly between species. The amino acid sequence of the humanANGIOSTATIN is substantially similar to the murine plasminogen fragment.But, it may be mentioned here that the active human ANGIOSTATIN sequencestarts either at the amino acid number 97 or 99 of an intact humanplasminogen amino acid sequence. In addition, human plasminogen haspotent anti-angiogenic activity even in a mouse tumor model. Thisexplains why both murine and human plasminogens andANGIOSTATIN/ENDOSTATIN molecules show fairly similar anti-angiogenicactivities in a variety of animal tumor models (3).

U.S. Pat. No. 5,792,845 issued on Aug. 11, 1998 to O'Reilly et al,teaches that therapies directed at control of the angiogenic processcould lead to the abrogation or mitigation of certain diseases. O'Reillyet al suggests that modulation of the formation of capillaries inangiogenic processes (such as wound healing and reproduction) is usefulsince undesired and uncontrolled angiogenesis can cause certain diseasesto progress. O'Reilly et al teaches that ANGIOSTATIN protein has thecapability of inhibiting angiogenesis, eg., to inhibit the growth ofbovine capillary endothelial cells in culture in the presence offibroblast growth factor.

U.S. Pat. No. 5,792,845 issued on Aug. 11, 1998 to O'Reilly et al,teaches that therapies directed at control of the angiogenic processcould lead to the abrogation or mitigation of certain diseases. O'Reillyet al suggests that modulation of the formation of capillaries inangiogenic processes (such as wound healing and reproduction) is usefulsince undesired and uncontrolled angiogenesis can cause certain diseasesto progress. O'Reilly et al teaches that ANGIOSTATIN protein has thecapability of inhibiting angiogenesis, e.g., to inhibit the growth ofbovine capillary endothelial cells in culture in the presence offibroblast growth factor.

U.S. Pat. No. 5,932,545 issued on Aug. 3, 1999 to Henkin et al teachesan anti-angiogenic drug in the form of a peptide or a salt thereof, totreat cancer, arthritis and retinopathy. The Henkin et al patent stateshowever that angiogenesis inhibitors could cause systemic toxicity inhumans. ANGIOSTATIN in the O'Reilly patent '845 is described and claimedas an Isolated nucleotide molecule with a specific sequence. It has beenstated however that the ANGIOSTATIN molecule as known at present is notsuitable for clinical trials.

ENDOSTATIN, which is also similar to ANGIOSTATIN, has been shown tocause a dramatic reduction of primary and metastatic tumors inexperimental animals. ENDOSTATIN is a 20 kDa C-terminal fragment ofcollagen XVIII. ENDOSTATIN could specifically inhibit endothelial cellproliferation and angio-genesis and thus, block tumor growth (2, 4).

It is important to note that ANGIOSTATIN is derived from plasminogen orplasmin. It has been shown that human prostate carcinoma cell linesexpress enzymatic activity that can generate bioactive ANGIOSTATIN frompurified human plasminogen or plasmin. This bioactive ANGIOSTATIN hasbeen shown to inhibit human endothelial cell proliferation, basicfibroblast growth factor-induced migration, endothelial cell tubeformation, and basic fibroblast growth factor-induced cornealangiogenesis. In an extension of this study, it was noted that a serineproteinase is necessary for ANGIOSTATIN generation (5).

ANGIOSTATIN, derived from plasminogen, selectively inhibits endothelialcell proliferation. When ANGIOSTATIN is given systemically it showspotent inhibitory action on the growth of tumor and renders metastaticand primary tumors to go into a dormant state by striking a balancebetween the rate of proliferation and apoptosis of the tumor cells (6).The very identification of ANGIOSTATIN has come from the observationthat when a primary tumor is present, the growth of metastases issuppressed. On the other hand, after tumor removal, the previouslydormant metastases develop new blood vessels (neovascularization) andgrow. Both serum and urine from the tumor-bearing animals, but not fromcontrols, showed very specific inhibitory action on the growth ofendothelial cells. Subsequent studies led to the purification of thisinhibitor of endothelial cells which was later identified as a 38 kDplasminogen fragment namely ANGIOSTATIN. It is now known thatANGIOSTATIN, which can also be obtained by a limited proteolyticdigestion of human plasminogen, but not intact plasminogen can beadministered systemically to block neovascularization and growth ofmetastases and primary tumors. A recombinant human ANGIOSTATIN whichcomprises of kringles 1-4 of human plasminogen (amino acids 93-470)expressed in Pichia pastoris has been prepared and is now available foruse. This recombinant ANGIOSTATIN showed the same physical properties asthat of the natural ANGIOSTATIN in terms of molecular size, binding tolysine, reactivity with antibody to kringles 1-3 (3, 7). Thisrecombinant ANGIOSTATIN when given to experimental animals, showedanti-angiogenic and anti-tumor activity (3). In addition, recombinantmouse ANGIOSTATIN was produced using the baculo-virus infected insectcells (8), which also (the secreted protein) showed potent inhibitoryaction on the proliferation of bovine capillary endothelial cells invitro. The conversion of plasminogen to ANGIOSTATIN by PC-3 cells is nowidentified to be due to two components released, urokinase (uPA) andfree sulfhydryl donors (FSDs). This is supported by the fact that evenin a cell-free system, ANGIOSTATIN can be generated from plasminogen byplasminogen activators (u-PA, tissue-type plasminogen activator, tPA orstreptokinase) in combination with any one of free sulfhydryl donorssuch as N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, orreduced glutathione. This cell-free derived ANGIOSTATIN also showedanti-angiogen activity both in vitro and in vivo and suppressed thegrowth of Lewis lung carcinoma metastases (9).

ANGIOSTATIN administration to mice with subcutaneoushemangioendothelioma and associated disseminated intravascularcoagulopathy revealed that in addition to a significant reduction in thesize of the tumor, increased survival, decrease in thrombocytopenia andanemia was noted (10). This indicates that ANGIOSTATIN may also beuseful to treat disseminated intravascular coagulopathy.

One of the mechanisms by which ANGIOSTATIN inhibits endothelial cellproliferation includes its ability to affect by 4 to 5 fold theexpression of E-selectin in proliferating endothelial cells (11). On theother hand, ANGIOSTATIN did not alter cell cycle progressionsignificantly. Further, ANGIOSTATIN also enhanced the adhesion activityin proliferating endothelial cells.

Rivas et al (12) studied the possible relationship between humanmacropahge metalloelastase (HME) expression, a member of the humanmatrix metalloproteinase family, which is believed to play an importantrole in ANGIOSTATIN generation, and ANGIOSTATIN production. Their studyshowed that patients whose tumors did not express HME mRNA and so didnot produce ANGIOSTATIN, had poorer survival than those whose tumorsshowed high expression of HME mRNA and ANGIOSTATIN generation. Thisstudy suggests that HME gene expression is closely associated withANGIOSTATIN generation and prognosis in patients with hepatocellularcarcinoma (HCC). This relationship between HME and ANGIOSTATIN isunderstandable since, metalloproteinase(s) can block angiogenesis byconverting plasminogen to ANGIOSTATIN (12,13,14).

Another mechanism by which recombinant human and murine ANGIOSTATINs canblock angiogenesis is by inducing apoptosis (programmed cell death) ofendothelial cells (15), similar to that seen with tumor necrosis factor(TNF) and transforming factor-beta 1 (TGF-beta1), which are also knownto induce apoptosis in endothelial cells.

Yet another mechanism by which ANGIOSTATIN can produce apoptosis andinhibit angiogenesis is probably by binding to ATP synthase. Using humanumbilical endothelial vein endothelial cells, Moser et al (16) observedthat ANGIOSTATIN bound in a concentration-dependent, saturable manner tothe alpha/beta sub-units of ATP synthase. This binding of ANGIOSTATIN tothe alpha/beta sub-unit of ATP synthase was inhibited by as much as 90%in the presence of anti-alpha-sub-unit ATP synthase antibody. Thisindicates that ANGIOSTATIN by binding to ATP synthase may actuallyshut-off ATP synthesis in the endothelial cells and this wouldeventually lead to death of the cells due to the non-availability ofATP, the main energy source for the survival of the cells. In addition,it was also reported that ANGIOSTATIN can inhibitextra-cellular-matrix-enhanced, t-PA catalysed plasminogen activation.This results in reduced invasive activity of endothelial cells (17). Allthese results indicate that ANGIOSTATIN has multiple actions by which itis able to block endothelial cell proliferation and angiogenesis.

Some of the factors which are known to inhibit the generation ofANGIOSTATIN include TGF-beta1 and plasminogen activator inhibitor type-1(PAI-1), at least, by human pancreatic cancer cells in vitro (18).

Twining et al (19) showed that plasmin, the active form of plasminogen,is necessary for the maintenance of normal cornea and for corneal woundhealing. It was also noted that plasmin is a major serine proteinase inthe human cornea and that cornea can synthesize plasminogen. Bothinterleukin-1alpha and 1 beta stimulated corneal plasminogen synthesisby almost 2 to 3fold where as interleukin-6 decreased cornealplasminogen synthesis by 40%. Thus, cornea seems to have the ability tosynthesize plasminogen, the precursor of plasmin and ANGIOSTATIN, andalso regulate its synthesis in response to injury and inflammation andIL-1 and IL-6 (19).

Though both ANGIOSTATIN and ENDOSTATIN and other similar anti-angiogenicmolecules provided an important therapeutic advance for cancertreatment, it should be emphasized here that the needed dosages of theseproteins, especially ANGIOSTATIN used in the animal studies seem to betoo high for clinical trials (20). Further, repeated injections andlong-term treatment with ANGIOSTATIN are required to obtain its maximalanti-tumor effect. In view of this, methods to supplement theanti-angiogenic action of ANGIOSTATIN and ENDOSTATIN and other similarcompounds are considered desirable. These methods include: use ofANGIOSTATIN along with other conventional anti-cancer drugs includingradiation and novel methods of delivery of ANGIOSTATIN to tumor cells(21). Mauceri et al (22) studied the combined effect of radiation withANGIOSTATIN and showed that this combination produced no increase intoxicity towards normal tissue. Both in vitro and in vivo studies showedthat these agents (radiation and ANGIOSTATIN) in combination target thetumor vasculature. In an extension of this study, Gorski et al (23)demonstrated that the efficacy of experimental radiation therapy ispotentiated by brief concomitant exposure of the tumor vasculature toANGIOSTATIN.

Two novel methods of delivery of ANGIOSTATIN and similar compounds tothe tumor cells that have been tried include:

-   -   (a) Nguyen et al (24) generated recombinant adeno-associated        virus (rAAV) vectors that carry genes encoding for ANGIOSTATIN,        ENDOSTATIN, and an antisense mRNA species against vascular        endothelial growth factor (VEGF). These rAAVs efficiently        transduced three human tumor cell lines that have been tested.        Further, testing of the conditioned media from cells transduced        with this rAAV or with rAAV-expressing ENDOSTATIN or ANGIOSTATIN        inhibited effectively endothelial cell proliferation in vitro.        These results indicate that rAAVs can be used to block        angiogenesis and cancer growth.    -   (b) In a different approach, Chen et al (25) examined whether        liposomes complexed to plasmids encoding ANGIOSTATIN or        ENDOSTATIN can inhibit angiogenesis and growth of tumors. These        studies revealed that plasmids expressing ANGIOSTATIN        (PCI-angio) or ENDOSTATIN (PCI-endo) can effectively reduce        angiogenesis and the size of the tumors implanted in the mammary        fat pad of male mice to a significant degree. In addition,        liposomes complexed to PCI-endo when given intravenously reduced        tumor growth in nude mice by nearly 40% when compared to        controls (25).

SUMMARY OF THE INVENTION

All the above factors and observations attest to the fact that malignanttumors are angiogenesis-dependent diseases. But, it should be mentionedhere that tumor-associated angiogenesis is a complex, multi-step processwhich can be controlled by both positive and negative factors. Itappears, as though, angiogenesis is necessary, but not sufficient, asthe single event for tumor growth (26). But, it is evident from severalexperimental results that angiogenesis may be a common pathway for tumorgrowth and progression. Though several anti-angiogenic agents are beingtried to arrest tumor growth, these are not without problems. Since themajority of these agents are proteins/peptides, their long-term use maylead to the development of antibodies which can neutralize their action.These anti-angiogenic substances need to be given repeatedly and some ofthem are unstable and are difficult to produce in large amounts.

In view of this, it is desirable and necessary to make efforts tostabilize and potentiate the actions of known anti-angiogenic molecules.

The present invention teaches the efficacious use of anti-angiogenicsubstances, which can inhibit endothelial cell proliferation andcoupling them to cis-unsaturated fatty acids, which also haveanti-angiogenic and cytotoxic actions on tumor cells, such that theactions of these substances are potentiated by each other. Further, asangiogenesis is involved in other disease processes such asinflammation, tumor metastasis, etc., it is envisaged that theconjugate(s) of anti-angiogenic substances and c-UFAs will be useful inthese diseases also.

In this context, it is important to note that the inventor has foundthat polyunsaturated fatty acids (PUFAs) such as gamma-linolenic acid(GLA), dihomo-GLA (DGLA), arachidonic acid (AA), eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA) can selectively kill the tumorcells ((27-32) and under specific conditions and in conjugation withsalts such as lithium and a lymphographic agent these fatty acids canactually behave as anti-angiogenic substances, i.e. they block all theblood supply to the tumor and also prevent generation of new bloodvessels. Using these fatty acids in this particular combination, theinventor has successfully treated human hepatocellular carcinoma andgiant cell tumor of bone with few or no side-effects.

Described hereinafter is a novel combination of a protein and a lipidand method(s) for its use. The protein referred to herein is a potentand specific inhibitor of endothelial proliferation and angiogenesis.The lipid may be one or more of the polyunsaturated fatty acids: LA(linoleic acid), GLA, DGLA, AA, ALA (alpha-linolenic acid), EPA, DHA andcis-parinaric acid. In this instance or method the polyunsaturated fattyacid needs to be given only once or at the most twice within a period of1 to 2 months. This invention teaches that unlikeANGIOSTATIN/ENDOSTATIN, these fatty acids are not only cytotoxic to thetumor cells but are also able to function as anti-angiogenic agents(33-35). Further, polyunsaturated fatty acids when given in theformulated form, are more potent than ANGIOSTATIN/ENDOSTATIN in theiranti-angiogenic and anti-cancer actions.

The invention in one aspect teaches a method of interrupting bloodsupply to a tumor region causing necrosis or apoptosis. The inventionalso provides a method of causing anti-angiogenic action in the tumorregion with the result that new blood vessels and collaterals are notformed to sustain the tumor. The present invention in another aspecttackles the issue of drug delivery to the target tissue and provides themost efficacious method of administering an admixture of selected PUFAswith other elements such as anti-angiogenic substances as will bedescribed hereinafter.

The invention in yet another aspect teaches a method of interruptingblood using a pre-determined admixture of at least a PUFA and ananti-angiogenic agent causing necrosis with very desirable results. Boththe PUFAs and anti-angiogenic compounds being similar in function, theinvention also provides a method of causing anti-angiogenic action inthe tumor region with the result that new blood vessels and collateralsare not formed to sustain the tumor in the tumor region treatedaccording to the invention. The present invention in another aspecttackles the issue of drug delivery to the target tissue and provides themost efficacious method of administering an admixture of selected PUFAsalong with an anti-angiogenic substance and other elements as will bedescribed hereinafter.

Tumor cells are deficient in phospholipase A2, an enzyme necessary forthe release of various PUFAs from the cell membrane lipids as a resultof which the production of anti-neoplastic PGs such as PGD2 are notelaborated. In addition, tumor cells secrete an excess of PGE2, animmunosuppressive and mutagenic substance. Further, tumor cells aredeficient in PUFAs such as GLA, AA, EPA and DHA due to the low activityof delta-6-desaturase. As a result of these metabolic changes, tumorcells are able to effectively circumvent body's defense and survive. Thepresent invention provides a method of causing necrosis of tumor cellsdespite their known survival pattern.

Anti-Cancer Actions of PUFAs

Tumor cells are not only deficient in PUFAs but also have low rate(s) oflipid peroxidation, contain relatively large amounts of antioxidantssuch as vitamin E and superoxide dismutase (SOD). It is also believedthat low rates of lipid peroxidation and consequent low amounts of lipidperoxides in the cells can contribute to an increase in the mitoticprocess which ultimately leads to an increase in cell proliferation.Thus, a deficiency of PUFAs, high amounts of antioxidants and thepresence of low amounts of lipid peroxides in the tumor cells cancontribute to the growth of tumor cells. This is supported by studies bythe inventor wherein it was noted that PUFAs such as GLA, DGLA, AA, EPAand DHA can decrease tumor cell proliferation. In addition, it was alsoobserved that when appropriate amounts of GLA, DGLA, AA, EPA and DHAwere administered to tumor cells and normal cells, obtained fromAmerican Type Culture Collection, only tumor cells were killed withouthaving any significant action on the survival of normal cells in vitro.In mixed culture experiments, in which both normal and tumor cells weregrown together, GLA showed more selective tumoricidal action compared toAA, EPA and DHA though, these latter fatty acids were also effective tosome extent. This indicated that selective delivery of GLA, DGLA, AA,EPA and DHA to tumor cells may offer a new therapeutic approach in thetreatment of cancer.

These in vitro results are supported by in vivo studies performed inanimal tumor models. For example, it was noted that GLA, DGLA, AA, EPAand DHA when used either in the form of pure fatty acid alone or in theform of fatty acid rich oils could inhibit the growth of skin papillomain mice, formation and growth of hepatoma in rats and ascitic tumorcells in the peritoneum of experimental animals. These results indicatethat these fatty acids can inhibit the growth of a variety of tumorseven in vivo. In further studies, it was noted that these fatty acidsare able to enhance free radical generation and the lipid peroxidationprocess selectively in the tumor cells but not so much in the normalcells and thus, are able to bring about their cancer killing action.

This ability of PUFAs to augment free radical generation and lipidperoxidation in the tumor cells is analogous to the anti-tumor action oflymphokines such as tumor necrosis factor (TNF) and interferon (IFN),both alpha and gamma varieties. These lymphokines (also referred to ascytokines) are capable of inducing the release of PUFAs from the cellmembrane lipid pool and enhance free radical generation in the cells.Similarly several anti-cancer drugs such as, but not limited to,doxorubicin and vincristine have the capacity to augment free radicalgeneration and promote lipid peroxidation. In addition, PUFAs and theirproducts can modulate immune response, augment a respiratory burst ofneutrophils and free radical generation by macrophages. This evidence isfurther testified by the observation that the incidence of cancer inEskimos is low as influenced by their traditional diet, which is rich inEPA and DHA. Inventor's studies have shown that PUFAs can be exploitedas possible anti-cancer agents either alone or in combination withlymphokines and traditional anti-cancer drugs.

In a series of investigations by the inventor, it was also observed thatthe cytotoxic action of anti-cancer drugs such as doxorubicin,vincristine and cis-platinum can be augmented by various PUFAs such asGLA, DGLA, AA, EPA and DHA. In addition, these fatty acids could alsoenhance the cellular uptake of these anti-cancer drugs by the tumorcells and thus, are able to potentiate the anti-cancer actions of thesedrugs. In another similar experiment by the inventor, it was alsoobserved that GLA, DGLA, AA, EPA and DHA were able to kill TNF resistantL-929 tumor cells in vitro. Further, these TNF-resistant tumor cellswere rendered TNF sensitive by prior treatment of these L-929 cells byGLA, DGLA, AA, EPA and DHA. These results indicate that PUFAs can notonly kill the tumor cells by themselves but are also capable ofpotentiating the cell killing effect of various anti-cancer drugs,lymphokines such as TNF and IFN and also render anti-cancer drug andTNF-resistant tumor cells sensitive to the cytotoxic action of variousanti-cancer drugs and lymphokines.

In another set of experiments, it was also noted that vincristineresistant tumor cells, KB^(chR)-8-5 (henceforth referred to as KB-8-5cells) can be made sensitive to the cytotoxic action of vincristine byGLA, DGLA, AA, EPA and DHA. Further, when sub-optimal doses ofvincristine and fatty acids were added together to these vincristineresistant cells produced optimal (i.e. significant) cell killing action.This shows that vincristine and other anti-cancer compounds and PUFAswhen added together to cancer cells, they potentiate the cytotoxicaction of each other. Fatty acid analysis of both vincristine sensitive(KB-3-1) and resistant (KB-8-5) cells revealed that the resistant cellshave low amounts of GLA, AA, EPA and DHA compared to the vincristinesensitive tumor cells indicating that a deficiency of these fatty acidsmay be responsible for their resistance to the cytotoxic actions ofanti-cancer drugs. Since, both vincristine sensitive and resistant tumorcells are easily (and to the same extent) killed by various PUFAs invitro, this demonstrates that even drug-resistant tumor cells can bekilled by these fatty acids.

In yet another set of experiments, the inventor also noted that L-929cells which are resistant to the cytotoxic action of tumor necrosisfactor (referred to as TNF-resistant L-929 cells) can also be madesensitive to the cytotoxic action of TNF by pre-treating these cellswith various PUFAs. In other words, L-929 cells which are resistant tothe cytotoxic action of TNF can be sensitized to the cytotoxic action ofTNF by PUFAs. This again indicates that PUFAs can not only kill thetumor cells but can also serve as sensitizing agents rendering varioustumor cells responsive to the cytotoxic action of various anti-cancerdrugs and lymphokines (cytokines) such as tumor necrosis factor.

It is to be noted in this context that PUFAs can bind to albumin andother proteins and hence, if given intravenously may not be available tobe taken up by the tumor cells and consequently may not be able to bringabout their cell killing action on the tumor cells. In view of this, itis desirable that PUFAs including GLA should be delivered to thepatients in such a manner that it is easily available to the tumor(tumor cells) and is delivered selectively to the tumor cells. It ishighly desirable that PUFAs including GLA be given intra-tumorally aswas experimentally done in the case of human gliomas, or,intra-arterially by selective intra-arterial infusion as was doneexperimentally in the case of hepatoma and giant cell tumor of the bone.But, it is also possible that in some cases of cancer such as Hodgkin'sand non-Hodgkin's lymphoma wherein the tumor cells are extremelysensitive to the cytotoxic actions of PUFAs, even oral administrationmay be sufficient as was observed in certain patients. Since, PUFAs canpotentiate the cell killing effect of anti-cancer drugs and lymphokines,it is desirable to administer a combination of PUFAs, anti-cancer drugs,lymphokines such as TNF and interferon or other anti-angiogenic agentsor a combination thereof with or without a carrier agent such as an oilylymphographic agent as the situation indicates. Further studies havealso revealed that PUFAs such as GLA, DGLA and EPA can prevent orameliorate the side effects of anti-cancer agents such asgamma-radiation and cis-platinum to the bone marrow cells of mice. Thus,it appears that when PUFAs and conventional anti-cancer drugs/agents aregiven together they not only potentiate the cytotoxic action of each onthe tumor cells and thus, produce a synergistic and/or additive actionin their ability to eliminate the tumor cells but it will also lead toelimination, reduction or amelioration of the side effects ofconventional anti-cancer agents. Since PUFAs are able to potentiate thecytotoxic action(s) of conventional anti-cancer agents and lymphokines,it is also possible that this will lead to a significant reduction inthe doses of these latter agents without compromising the ultimatebenefit namely, elimination of tumor cells or the tumor.

Some of the phenomena which reduce the efficacy of the cytotoxic actionof PUFAs and conventional anti-cancer drugs/agents in vivo as comparedto in vitro results include the following:

-   -   a. PUFAs when administered orally or intravenously can bind to        albumin and other proteins in living beings and may not be        available to be taken up by the tumor cells. But this ability of        PUFAs to bind to proteins is made use of in the present        invention and is detailed below.    -   b. The cytotoxic action of PUFAs is produced by the augmentation        of free radical generation and lipid peroxidation in only tumor        cells (but not in normal cells). The intensity of the cytotoxic        action is disadvantageously reduced in actual clinical efforts        because of inefficient transportation of the fatty acids to the        target areas.    -   c. Continued blood supply to tissue with proliferative cell        disorders is not conducive to bringing about a significant        amount of necrosis especially if the malignant cells multiply        faster than they are being destroyed.    -   d. It was found from a study reported in a June, 1994 “Cancer        letters” publication authored by N. Madhavi and U. N. Das that        antioxidants like vitamin E and the superoxide anion quencher,        superoxide dismutase (SOD) could completely inhibit free radical        generation and lipid peroxidation generated by PUFAs like GLA,        EPA and DHA. It appears that selective drug delivery to the        target tissue will be conducive to the efficacy of the        beneficial action of the PUFAs.

The present invention in one aspect resides in a method of inhibitingblood supply to a tumor by using two types of substances: one a lipidand the other a protein or a peptide both of which have very potentanti-angiogenic action. In addition, the invention also comprises of thesteps of: locating an artery which carries major blood supply to thetumor, said artery being one that is proximate to the tumor, andintra-arterially injecting into the located artery a predeterminedquantity of a polyunsaturated fatty acid (PUFA) in the form of asolution of at least one PUFA chosen from LA, GLA, DGLA, AA, ALA, EPA,DHA and cis-parinaric acid in combination with a protein/peptide withanti-angiogenic substance(s).

The invention in another aspect resides in a method for treating tumorsand for facilitating visualization of remission of the tumor in responseto treatment, comprising the steps of:

-   -   (a) locating an artery which carries a major portion of blood        supply to the tumor and is adjacent to the tumor;    -   (b) obtaining an initial radiographic image of the tumor region;    -   (c) injecting into the artery a mixture of (i) an oily        lymphographic agent, (ii) a lithium salt solution of at least        one PUFA chosen from LA, GLA, DGLA, AA, ALA, EPA, DHA; and        cis-parinaric acid (iii) an anti-angiogenic protein/substance        which is co-valently linked to the fatty acid or form a mixture        (fatty acid+anti-angiogenic protein or peptide).    -   (d) obtaining second and subsequent radiographic images of the        tumor regions after predetermined lapses of time; and comparing        the initial radiographic images with the second and subsequent        radiographic images to assess the extent of remission of the        tumor.

The invention in another aspect resides in a method of causing necrosisin a cancerous tumor by inhibiting blood supply to the tumor, and alsoby direct cytotoxicity to the tumor cells, comprising the steps of: (a)locating an artery proximate to the tumor which carries major bloodsupply to the tumor; (b) injecting into the located artery a mixture of(i) an anti-angiogenic protein/peptide; (ii) a lithium salt solution ofat least one essential fatty acid chosen from LA, GLA, DGLA, AA, ALA,EPA, DHA and cis-parinaric acid; (c) waiting for a predetermined timeperiod and assessing a degree of necrosis in the tumor by examining by aradiographic study or by other means; and, (d) repeating step (b) ifnecessary to increase the necrosis.

In yet another aspect, the invention resides in a method of treating aglioma and visualizing remission of the glioma as it responds totreatment, comprising:

-   -   (a) obtaining an initial radiographic image of a region        containing the glioma;    -   (b) injecting into the glioma region an admixture of (i) a        sodium salt or any other suitable salt solution of at least one        polyunsaturated fatty acid chosen from LA, GLA, DGLA, AA, ALA,        EPA, DHA and cis-parinaric acid or a combination there of along        with an anti-angiogenic protein/peptide;    -   (c) obtaining second and subsequent radiographic images of the        glioma region after predetermined lapses of time; and comparing        the initial radiographic pictures which shows the glioma, with        second and subsequent radiographic images of the glioma region        to visualize and assess the extent of remission of the glioma.

In yet another aspect, the invention resides in a method of treatingmammalian cell proliferative disorders using an emulsion of a lithiumsalt of a PUFA or combinations of PUFAs and a predeterminedanti-angiogenic protein/peptide administered parenterally including asubcutaneous route. Preferably, the intra-arterial administration of theadmixture containing PUFA(s) is done through a catheter. Also, theartery carrying major blood supply to the tumor is to be understoodherein as synonymous to the artery which will supply the tumor feedingvessels. Owing to a phenomenon which is consequent to inhibiting bloodsupply, the present invention makes it not conducive to the formation ofnew blood vessels i.e. angiogenesis. The anti-angiogenic protein indifferent implementations of this invention may be ENDOSTATIN orANGIOSTATIN or any other anti-angiogenic substance.

DETAILED DESCRIPTION

Essential fatty acids are precursors of eicosanoids and are importantstructural components of cell membranes. They also provide thesubstrates for the generation of lipid peroxidation products which havean inhibitory action on cell proliferation. Tumor cells are known tohave low delta-6-desaturase activity, an enzyme necessary for thedesaturation of dietary linoleic acid (LA, 18:2, n-6) andalpha-linolenic acid (ALA, 18:3, n-3) to their respective products. Inan earlier study, the inventor has shown that hepatocarcinogens,diethylnitrosamine (DEN) and 2-acetylaminofluorine (2-AAF), can suppressthe activity of delta-6-desaturase and delta-5-desaturase resulting inlow levels of gamma-linolenic acid (GLA, 18:3, n-6) and arachidonic acid(AA, 20:4, n-6) and eicosapentaenoic acid (EPA, 20:5, n-3) anddocosahexaenoic acid (DHA, 22:6, n-3) in the tumor cells. These resultsled the inventor and others to study the effect of various fatty acidson the survival of tumor cells in vitro. Addition of EFAs (LA and ALA)and other PUFAs such as GLA, DGLA, AA, EPA, DHA and cis-parinaric acidto a variety of tumor cells in vitro showed that only tumor cells arekilled by these fatty acids without harming the normal cells. Thisselective tumoricidal action of fatty acids seems to be mediated by freeradicals and lipid peroxides. Similar to these fatty acids, radiation,some anti-cancer drugs and cytokines (lymphokines) also seem to have theability to generate free radicals in tumor cells and thus, bring abouttheir tumoricidal actions.

Since drug resistance is a major obstacle in the clinical treatment ofcancer and as PUFAs have selective tumoricidal action, the inventorstudied the effects of PUFAs on drug-resistant tumor cells and theirmodulating influence on the actions of anti-cancer drugs. In the abovecontext, in addition to producing reversal of tumor cell drug resistanceby the administration of polyunsaturated fatty acids, it is seen fromthe invention that the manner of targeting the cancerous tissue is verycritical to the efficacy and the speed with which necrosis can bebrought about. More particularly, it is realized through this inventionthat by delivering a chosen admixture of salts of predeterminedpolyunsaturated fatty acids and predetermined anti-angiogenicsubstance(s) to the tumor site intra-arterially, intra-venously,subcutaneously, intra-peritoneally or by direct injection into the tumorbed, a very beneficial and hitherto unknown effect in terms ofinhibiting blood supply to the tumor site and inducing tumor cell lysisis achieved simultaneously.

In clinical studies conducted by the inventor with PUFAs, the inhibitionof blood supply was pronounced enough to cause cutting off blood supplyto the tumor site with very little time lag. In other instances, anunmistaken strangling of blood supply to the tumor region was observed,but was relatively gradual.

One aspect of the invention consists in the preparation of acombination/composition of treatment of cancer in which one or more ofLA, GLA, DGLA, AA, ALA, EPA, DHA and cis-parinaric acid are administeredwith conventional anti-cancer agents/drugs including anti-angiogenicprotein/peptide with or without an oily lymphographic agent or any othersuitable agent for the delivery of these compounds; optionally,radiation may be included. The PUFAs may be provided in a daily dose of0.5 mg to 50 gm together with appropriate doses of conventionalanti-cancer drugs such as vincristine, doxorubicin, L-asparaginase,cis-platinum, busulfan, etc., in a daily/weekly/monthly dose of 1 mg to50 gm depending on the requirement and the stage of the disease and asmay be determined from time to time with or without the addition ofanti-angiogenic protein/peptide such as ANGIOSTATIN/ENDOSTATIN in a doseof 1 mg to 100 mg/kg of body weight per day. The word anti-angiogenicsubstance is understood as one or more of the following substances:ANGIOSTATIN, ENDOSTATIN, platelet factor-4, TNP-470, thalidomide,interleukin-12, metalloprotease inhibitors (MMP), anti-adhesionmolecules (in their desired dose). The combination of PUFAs,conventional anti-cancer drugs, anti-angiogenic substances and the oilylymphographic agent may be administered by any one or different routesat the same time or at different times and intervals by selecting anappropriate route for each administration or in combination, e.g., oral,parenteral including intra-arterial infusion, intravenous, subcutaneous,intra-peritoneal, topical, anal, vaginal routes as suppositories, orlocal injection directly into the tumor bed under the guidance ofappropriate equipment such as but not limited to radiological guidance(X-rays), CT guidance or MRI guidance or by stereostaxic guidance. Thedaily dose(s) of these compounds may not exclude the administration oflong acting preparations or depot preparation once or more times in aday, week, month or at some other appropriate time interval asdetermined from time to time depending on the necessity. The fatty acids(PUFAs) may be present in any physiologically acceptable form includingbut not limited to glycerides, esters, free acids, amides, phospholipidsor salts. The conventional anti-cancer drugs may be administered bythemselves or in conjugation with PUFAs (either alone or in combinationsuch as GLA alone or GLA+AA, LA, DGLA, ALA, EPA or DHA). Similarly theanti-angiogenic substance(s) may be given by themselves or inconjugation with PUFAs. For intra-arterial infusion orintravenous/subcutaneous injection/infusion or administration of LA,GLA, DGLA, AA, ALA, EPA, DHA and/or cis-parinaric acid these may begiven by themselves or in combination or dissolved or conjugated in/withanti-angiogenic substances and in any other suitable solution that canbe given parenterally but not limited to them. All these PUFAs,conventional anti-cancer drugs, anti-angiogenic substances andlymphographic agent may each be given alone or in combination thereof orall together or separately at the same time or at different timeintervals on the same day/week/month either by same route or differentroutes as the situation demands.

In order to observe or ascertain and record progress made in patientsafter administration of admixture according to this invention, images ofthe affected area e.g., tumor region before and after treatment can beobtained by various known modalities such as computerized axialtomography (CT), magnetic resonance imaging (MRI), etc.

EXAMPLES

(1) Hard (wherein the PUFAs have been microencapsulated) or soft gelatincapsules (wherein the fatty acids are present in an oily form) made byaccepted normal or forms or methods and are administered to personssuffering from cancer in conjunction with conventional anti-cancer drugsand/or anti-angiogenic substances in the doses as stated supra.

(2) Hard or soft gelatin capsules made by conventional methods, in whichthe fatty acids, the anti-cancer drugs and anti-angiogenic substancesre-incorporated together in the same capsule and are administered topersons suffering from cancer.

(3) As intra-tumoral preparation in appropriate doses (from 0.5 mg to 50mg per day) of pure LA, GLA, DGLA, AA, ALA, EPA and DHA eitherindividually or in combination thereof especially with anti-angiogenicsubstances for the treatment of human brain gliomas or any otheraccessible tumor (e.g., urinary bladder cancer, carcinoma of theesophagus, carcinoma of the lung, breast cancer etc.) by any route byusing flexible fiber optic scopes such as bronchoscope, urethroscope,hysteroscope, etc. In the case of tumors of the head and neck the fattyacids are administered either by direct intra-tumoral route or byselective catheterization of the tumor feeding vessel(s) either byfemoral, brachial or carotid routes or by subcutaneous route orintravenous route. The PUFAs and anti-angiogenic substances can be givento these patients daily, weekly or monthly or as and when necessarydepending on the requirement and response of the patient to thetreatment.

(4) Administered as selective intra-arterial infusion or injection intothe tumor feeding vessel by femoral, brachial or carotid routes or anyother suitable route or in a combination thereof the PUFAs either aloneor in combination with anti-cancer drugs/anti-angiogenic substances withor without the oily lymphographic agent or any other suitable agent allin a mixture or in conjugated form(s) (like GLA+any conventionalanti-cancer drug or drugs+anti-angiogenic substance,LA/GLA/DGLA/AA/ALA/EPA/DHA/cis-parinaric acid all individually or incombination thereof+conventional anti-cancer drug(s)+anti-angiogenicsubstance(s)+lymphographic agent,LA/GLA/DGLA/AA/ALA/EPA/DHA/cis-parinaric acid in combination with orconjugated to anti-angiogenic substance(s) or emulsified with or mixedwith oily lymphographic agent, LA/GLA/DGLA/AA/ALA/EPA/DHA/cis-parinaricacid alone or in combination thereof in oily lymphographic agent as amixture or emulsion or as a conjugate(s) and a variety of othercombinations thereof. This preparation may be administered daily, weeklyor monthly or at some other appropriate time interval.

(5) Topical preparation of PUFAs either alone or in combination thereofwith conventional anti-cancer drugs or anti-angiogenic substance(s) in asuitable delivery vehicle in which daily doses (ranging from 0.5 μg to100 mg) are applied to primary skin cancers including Kaposi's sarcomalocally and/or conventional anti-cancer drugs are given either orally orparenterally.

By the different embodiments of the invention method described supra, itbecomes known that:

-   -   (i) when PUFAs or cis-EFAs (essential fatty acids described here        are also called as cis-fatty acids as by virtue of their        structure are referred to as cis-EFAs as they are in        cis-configuration) are administered to patients intra-arterially        or even otherwise as a combination with anti-angiogenic        substance(s), there are less chances of albumin and other        proteins binding to the fatty acids. Consequently, PUFAs thus        administered using the invention are better available to be        taken up by the tumor cells.    -   (ii) Owing to the efficient transportation of PUFAs to the tumor        site as described hereinbefore, there is increased intensity of        the cytotoxic action of PUFAs and the administered anti-cancer        agents (drugs or anti-angiogenic substance(s) or a combination        thereof). Thus, using the invention, there is relatively better        augmentation of free radical generation and lipid peroxidation        in the tumor cells, thereby facilitating a greater degree of        necrosis.    -   (iii) Inhibiting blood supply to the tumor region by the method        of invention prevents cell proliferation in the tumor region,        thus enabling healthy tissue to to grow back into place.    -   (iv) The inhibition otherwise caused by vitamin E and superoxide        dismutase to free radical generation and lipid peroxidation        produced by PUFAs, is reduced in the method of this invention        because of the manner of transportation of PUFAs to the tumor        site in combination with anti-angiogenic substance(s)        intra-arterially through a proximate artery or intravenously or        subcutaneously.

It is also within the purview of this invention, as stated supra toadminister an admixture of PUFAs, anti-cancer drugs, and selectedanti-angiogenic substance(s) at the same time, administeringpredetermined doses of PUFAs orally. All such variations are envisagedto be within the ambit of this invention.

Application to mammals: Even though the examples described supra relateto humans, it is envisaged that the method of inhibiting blood supplyand using admixture of this invention including an anti-angiogenicsubstance are equally applicable to other mammals.

EQUIVALENTS

While this invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Also sodium and potassiumsalts are considered equivalents of each other. Imaging techniquesreferred to herein are intended to include CAT, MRI, X-rays and otherpossible imaging methods. Those skilled in the art will recognize or beable to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedspecifically herein. Such equivalents are intended to be encompassed inthe scope of the appended claims.

REFERENCES

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1. A method of inhibiting blood supply to a tumor, comprising: (a)Locating an artery which carries major blood supply to the tumor, saidartery being one that is proximate to the tumor; and (b)Intra-arterially injecting into located artery a predetermined quantityof one or more anti-angiogenic substance(s), and a predeterminedquantity of a salt of at least one polyunsaturated fatty acid chosenfrom linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid,arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid,docosahexaenoic acid, and cis-parinaric acid.
 2. A method as in claim 1wherein said polyunsaturated fatty acid is in the form of a lithium saltsolution and wherein said fatty acid is in a range of 0.5 mg to 50 gm.3. A method as in claim 1 wherein said (b) comprises intra-arteriallyinjecting said predetermined quantity of the polyunsaturated fatty acidand in the form of a lithium salt solution of a polyunsaturated fattyacid, wherein said anti-angiogenic substances is provided in an amountof 1 to 1000 mg/kg/body weight, said solution of polyunsaturated fattyacid further comprising a substance chosen from glycerides, esters, freeacids, amides, phospholipids, and salts.
 4. A method as in claim 1,wherein the polyunsaturated fatty acid is in the form of a lithium saltsolution containing said anti-angiogenic substance(s) and an anti-cancerdrug predetermined quantity of said anti-angiogenic substance chosenfrom: an anti-angiogenic substance naturally occurring as a protein,platelet factor-4, TNP-470, thalidomide, interleukin-12, andmetalloprotease inhibitors, and a predetermined anti-cancer drug.
 5. Amethod of treating a tumor and facilitating visualization of remissionof the tumor response to treatment, comprising: (a) Locating an arterywhich carries a major portion of blood supply to said tumor and isadjacent to the tumor; (b) Obtaining an initial radiographic image oftumor; (c) Injecting into located artery a mixture of at least (i) anoily lymphographic agent as a carrier containing one or more ofanti-angiogenic substance(s) and (ii) a lithium salt solution of atleast one polyunsaturated fatty acid chosen from linoleic acid,gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid,alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, andcis-parinaric acid (d) obtaining second and subsequent radiographicimages of the tumor after predetermined lapses of time; and (e)comparing the initial radiographic image with the second and thesubsequent images to assess an extent of said remission of the tumor. 6.A method as in clam 5 wherein step (c) comprises intra-arteriallyinjecting said mixture containing, causing anti-angiogenic action byinhibiting the blood supply to the tumor, wherein further the oilylymphographic agent acts as a carrier for said lithium salt solution ofpredetermined quantities of said polyunsaturated fatty acids.
 7. Amethod of treating a cancerous tumor, comprising (a) using an oilylymphographic agent as a carrier for (i) at least one polyunsaturatedfatty acid chosen from a lithium salt of at least one of linoleic acid,gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid,alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, andcis-parinaric acid; and (ii) a predetermined anti-cancer drug, andanti-angiogenic substances mixed with the polyunsaturated fatty acids orcoupled with fatty acids; and (c) administering by injecting into saidcancerous tumor a predetermined quantity of the fatty acids, theanti-cancer drug and predetermined said anti-angiogenic substance in theoily lymphographic agent as a carrier.